Angiotensin converting enzyme (peptidyldipeptide hydrolase, hereinafter referred to as ACE) occupies a central role in the physiology of hypertension. The enzyme is capable of converting the decapeptide angiotensin I, having the sequence EQU AspArgValTyrlleHisProPheHisLeu
to an octapeptide, angiotensin II by removal of the carboxyterminal HisLeu. The symbols for various chemical entities are explained in the following table:
Ala=L-alanine PA0 Arg=L-arginine PA0 Asp=L-aspartic acid PA0 &lt;Glu=pyro-L-glutamic acid PA0 Gly=glycine PA0 Hip=Hippuric acid (Benzoyl glycine) PA0 His=L-histidine PA0 Ile=L-isoleucine PA0 Leu=L-leucine PA0 Phe=L-phenylalanine PA0 Pro=L-proline PA0 Ser=L-serine PA0 Trp=L-tryptophan PA0 Tyr=L-tyrosine PA0 Val=L-valine PA0 .gamma.-Glu=L-glutamic acid residue in peptide linkage at the .gamma.-carboxyl group PA0 ACE=Angiotensin converting enzyme PA0 Hepes=N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid
Angiotensin I is formed by the action of the enzyme renin, an endopeptidase found in kidney, other tissues and plasma, acting on a serum .alpha.-2 globulin.
Blood pressure is affected by certain peptides found in the blood. One of these, angiotensin II, is a powerful pressor (blood pressure elevating) agent. Another, bradykinin, a nonapeptide with the sequence ArgProProGlyPheSerProPheArg is a powerful depressor (blood pressure lowering) agent. In addition to a direct presson effect, angiotensin II stimulates release of aldosterone which tends to elevate blood pressure by causing retention of extracellular salt and fluids. Angiotensin II is found in measurable amount in the blood of normal humans. However, it is found at elevated concentrations in the blood of patients with renal hypertension.
The level of ACE activity is ordinarily in excess, in both normal and hypertensive humans, of the amount needed to maintain observed levels of angiotensin II. However, it has been found that significant blood pressure lowering is achieved in hypertensive patients by treatment with ACE inhibitors. [Gavras, I., and Vukovich, R. A., New Engl. J. Med. 291, 817 (1974)].
ACE is a peptidyldipeptide hydrolase. It catalyzes the hydrolysis of the penultimate peptide bond at the C-terminal end of a variety of acylated tripeptides and larger polypeptides having an unblocked .alpha.-carboxy group. The peptide hydrolysis is represented diagramatically as: R--A.sub.2 --A.sub.1 +H.sub.2 O.fwdarw.R--OH+H--A.sub.2 --A.sub.1, wherein A.sub.1 is an amino acid at the carboxyl terminus of the peptide, A.sub.2 is an amino acid linked to A.sub.1 by a peptide bond, R is an N-substituted amino acid linked to A.sub.2 by a peptide bond. The action of ACE results in hydrolytic cleavage of the penultimate peptide bond from the carboxyl-terminal end yielding as reaction products a dipeptide, HA.sub.2 A.sub.1, and a remnant, R--OH.
The reactivity of the enzyme varies markedly depending on the substrate. At least one type of peptide bond, having the nitrogen supplied by proline, is not hydrolyzed at all. The apparent Michaelis constant (Km) varies from substrate to substrate over several orders of magnitude. For general discussion of the kinetic parameters of enzyme catalyzed reactions, see Lehninger, A., Biochemistry, Worth Publishers, Inc., New York, 1970, pp. 153-157. Many peptides which are called inhibitors of the enzymatic conversion of angiotensin I to angiotensin II are in fact substrates having a lower Km than angiotensin I. Such peptides are more properly termed competitive substrates. Examples of competitive substrates include bradykinin, and the peptide BPP.sub.5.varies. (also called SQ20475) from snake venom, whose sequence is &lt;GluLysTrpAlaPro.
Numerous synthetic peptide derivatives have been shown to be ACE inhibitors by Ondetti, et al. in U.S. Pat. No. 3,832,337 issued Aug. 27, 1974.
The role of ACE in the pathogenesis of hypertension has prompted a search for inhibitors of the enzyme that could act as antihypertensive drugs. See for example U.S. Pat. Nos. 3,891,616, 3,947,575, 4,052,511 and 4,053,651. A highly effective inhibitor, with high biological activity when orally administered, is D-3-mercapto-2-methylpropanoyl-L-proline, designated SQ14225, disclosed in U.S. Pat. No. 4,046,889 to Ondetti et al., issued Sept. 6, 1977, and in scientific articles by Cushman, D. W. et al., Biochemistry 16 5484 (1977), and by Ondetti, M. et al., Science, 196 441 (1977). The inhibitor SQ14225 reportedly has an I.sub.50 value of 2.3.times.10.sup.-8 M. The I.sub.50 value is defined as a concentration of inhibitor required to produce 50% inhibition of the enzyme under a standard assay system containing an approximately K.sub.m level of substrate.
The mode of action of SQ14225 has been based upon a model of the active site of ACE developed by analogy with the berter known related enzyme, carboxypeptidase A. The active site was hypothesized to have a cationic site for binding the carboxyl end group of the substrate and a pocket or cleft capable of binding the side chain of the C-terminal amino acid and providing especially tight binding for the heterocyclic ring of a terminal proline residue. A similar pocket for the penultimate amino acid residue was postulated, and the published data suggested a rather stringent steric requirement, since the D-form of the inhibitor was substantially more potent than its stereoisomer or the 3-methyl and unsubstituted analogs. The sulfhydryl group on the inhibitor, postulated to be bound at the active site near the catalytic center, was believed to play a central role in inactivation of the enzyme by combining with the zinc moiety known to be essential for catalytic activity. Substituents on the sulfhydryl, such as a methyl group, and an S-acetyl derivative, substantially reduced potency of the inhibitor. See Cushman, D. W., et al., Biochemistry, supra.
In vitro study of the mechanism by which SQ14,225 and its analogs act to inhibit ACE has been somewhat hampered by the instability of these molecules under ambient conditions. For example, it has been observed that a fresh aqueous solution of concentration, e.g., 1 mg per ml of SQ14,225 at a pH of about 8 becomes substantially less active upon standing for as little as 30 minutes, and that activity continues to decrease as the solution stands for longer periods. It is believed that this loss in activity is mainly the result of dimerization of SQ14,225 occurring at the sulfhydryl end groups, whereby a disulfide is formed which is largely inactive as an inhibitor. Since the free sulfhydryl group is highly reactive and may be readily oxidized to polar acidic moieties such as sulfone and sulfoxide groups, it may also be that the observed in vitro loss of activity of aqueous solutions of SQ14,225 on standing is in some part a consequence of one or more such oxidation reactions, with formation of a sulfone or sulfoxide which does not function effectively as an inhibitor for ACE.
Such reports of SQ14,225 clinical testing as are currently available, some of which refer to the compound under the name "Captopril", suggest that the product is sufficiently stable in the normal gastric and intestinal environments of most patients to be an effective inhibitor for ACE when administered orally. It is not yet clear, however, whether there may be a group of patients for which SQ14,225 is substantially ineffective. Because of the high reactivity of the free sulfhydryl group, SQ14,225 could readily form mixed disulfides with serum, cellular proteins, peptides or other free sulfhydryl group-containing substances in the gastric or intestinal environments, in addition to the possibility for dimer formation or oxidative degradation reactions. A mixed disulfide with protein may be antigenic and, indeed, occasional allergic reactions have been clinically observed. See Gavras, et al., New England J. Med. 298, 991 (1978). Disulfides and oxidative degradation products of SQ14,225, if formed, may at best be expected to be largely ineffective as inhibitors. It may be hypothesized accordingly that dose response to SQ14,225 may vary with conditions of administration and among individual patients. Moreover, in at least some patients, unwanted side effects may occur or maintenance of an effective concentration of the inhibitor in the body may be difficult to control.
Thiolester compounds generally are thought to be highly reactive in that the thiolester linkage is readily hydrolyzable to a sulfhydryl moiety and a carboxylic moiety. Thiolesters are accordingly often used as active ester intermediates for acylation under mild conditions. Such groups as, e.g., acetylthio have been used as blocking groups in the above cited Ondetti, et al. patents. Thiolester intermediates are also postulated to occur in the biosynthesis of cyclic peptides such as tyrocidin or gramicidin S. See Lipmann, F. in Accounts Chem. Res. 6, 361 (1973).
It is accordingly of particular significance that the inhibitors of this invention, albeit thiolesters, appear to be exceptionally stable in vitro and relatively inert to oxidation. For example, it has been observed that solutions at approximately 1 mg per ml concentration, of an inhibitor of this invention, at pH levels of from about 7 to about 9.5 at room temperature in the presence of sodium bicarbonate give no color response to a selective staining agent for free sulfhydryl groups even after standing for two hours, thereby indicating stability to hydrolysis of the thiolester linkage.
Because of this observed in vitro stability, it is ized that the inhibitors of this invention may be effective in environments where SQ14,225 is largely ineffective or may be capable of administration under less rigorously controlled conditions.